Requirements for cell surface expression of the influenza hemagglutinin (HA) were studied using a recombinant of SV40 which had full-length DNA sequences coding for the influenza virion HA inserted into the late region of SV40 DNA and propagated in the presence of tsA SV40 helper. Infection of primate cells with the SV40-HA recombinant produced a functional glycosylated HA polypeptide that accumulated on the cell surface. To delineate the protein domains necessary for surface expression of the HA polypeptide, mutations were engineered in the recombinant SV40-HA DNA. One mutant of interest sustained a deletion of 5 base pairs at the Bam HI site generating a shift in reading frame for the hydrophobic sequence of 24 amino acids as well as the C' terminal 13 amino acids. This shift yielded a terminal sequence in which 21 of 24 amino acids were hydrophobic or neutral followed by 4 charged amino acids. Although its C' terminus resembled wild-type topologically, the mutant HA was not secreted, was not expressed on the cell surface, and was only present intracellularly. These findings suggest a role for the hydrophobic COOH-terminus not only in cell surface expression but in secretion related glycosylation.